Objective: To determine the etiology of first time urinary tract infection (UTI) among children presenting at a tertiary care hospital of Karachi, Pakistan. Study Design: Cross-sectional study. Setting: Department of Pediatrics, National Institute of Child Health, Karachi. Period: December 2022 to May 2023. Methods: Children of either gender, aged between 2 months to 12 years, and having UTI for the first time were analyzed. Medical history was obtained and socio-demographic as well as clinical characteristics were noted. Relevant clinical evaluation, laboratory parameters, ultrasonographic findings and etiological pattern were noted. Results: In a total of 138 children, 84 (60.9%) were girls. The mean age was noted to be 3.42±2.41 years. The mean duration of symptoms was 11.51±4.27 days. The most frequent presenting symptoms were fever, painful micturation, and abdominal pain, reported by 105 (76.1%), 88 (63.8%), and 64 (46.4%), respectively. The most frequent structural renal abnormalities were internal echos in bladder, unilateral mild hydronephrosis, and bilaterial moderate hydronephrosis, noted in 38 (27.5%), 15 (10.9%), and 14 (10.1%) children, respectively. Escherichia coli, Klebsiella spp., Pseudomonas spp., and others found in 74 (53.6%), 36 (26.1%), 22 (15.9%), and 6 (4.3%), respectively. Conclusion: The microbial analysis demonstrated a predominant role of Escherichia coli, followed by Klebsiella spp. and Pseudomonas spp. in causing UTIs for the first time in children.